The goal of our studies is to elucidate the biochemical mechanism of fatty acylation of secreted proteins. Our focus is on Sonic hedgehog (Shh) and Wnts, signaling proteins that play key roles during embryonic development and tumorigenesis. In order to signal, Shh must be modified by attachment of palmitate, a reaction catalyzed by Hhat (hedgehog acyltransferase). Hhat is a member of the MBOAT (membrane bound O-acyltransferase) family of multipass membrane proteins. We developed cell-based and in vitro assays for Shh palmitoylation, purified Hhat and performed high throughput screening to identify small molecule inhibitors of Hhat. Hhat inhibitors block the growth of human pancreatic and breast cancer cells and are currently undergoing development as potential chemotherapeutics. Parallel studies have explored fatty acylation of Wnt proteins. Wnts are modified by the MBOAT acyltransferase Porcupine (Porcn) with palmitoleic acid, an unusual lipid modification required for production of an active Wnt signal. Although we have working models for Hhat and Porcn-mediated lipid modification, critical gaps remain in our knowledge of how secreted morphogens are modified by distinct fatty acyltransferases. We aim to use in silico and empirical methods to generate 2D and 3D structural models for Hhat and Porcn, and use this as a basis for revealing how MBOAT enzymes recognize their fatty acid and protein substrates. Aim 1. Structure, function and regulation of Hhat. In collaboration with Chris Sander, we will use the EVfold_membrane algorithm to predict the 3D structure of Hhat in silico and test the model empirically. Substrates and/or proteins that bind to predicted cytoplasmic and intralumenal loops of Hhat will be identified using GST pulldown experiments. Regulation of Hhat by posttranslational modification, amplification and/or mutation in normal and cancer cells will be explored. Aim 2. How are substrates delivered to and recognized by Hhat? It is not known how palmitoyl CoA, which is not permeable across biological membranes, is delivered to Hhat in the ER lumen. We will determine whether palmitoyl CoA enters the ER lumen through a carnitine-dependent system, and if Hhat regulates this process. A proteomics-based approach will be used to identify additional Hhat substrates by combining click chemistry with SILAC (stable isotope labeling with amino acids in cell culture). Newly identified Hhat substrates will be analyzed for their roles in regulating breast cancer cell growth. Aim 3. To decipher the molecular mechanism of Porcn-mediated Wnt acylation. We will test the hypothesis that Porcn recognizes the Serine acylation site within Wnt in the context of a disulfide- linked structure by establishing an in vitro Wnt acylation assay with protein and peptide substrates. Porcn will be purified and its enzymologic parameters will be characterized. A transmembrane topology map of Porcn will be generated using differential membrane permeabilization and protease protection assays.